Glutathione and ascorbic acid protect Arabidopsis plants against detrimental effects of iron deficiency

LeonorRamrez 1 Carlos GuillermoBartoli 0 LorenzoLamattina 1 0 Instituto de Fisiologa Vegetal , Facultad de Ciencias Naturales, Universidad Nacional de La Plata-CCT La Plata CONICET , CC 327, 1900, La Plata, Argentina 1 Instituto de Investigaciones Biolgicas , UE-CONICET-UNMdP, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata , Funes 3250, CC 1245, 7600, Mar del Plata, Argentina Iron is an essential micronutrient required for a wide variety of cellular functions in plant growth and development. Chlorosis is the first visible symptom in iron-deficient plants. Glutathione (GSH) and ascorbic acid (ASC) are multifunctional metabolites playing important roles in redox balancing. In this work, it was shown that GSH and ASC treatment prevented chlorosis and the accumulation of reactive oxygen species induced by iron deficiency in Arabidopsis leaves. In iron deficiency, GSH and ASC increased the activity of the heme protein ascorbate peroxidase at a similar level to that found in iron-sufficient seedlings. GSH was also able to preserve the levels of the iron-sulfur protein ferredoxin 2.GSH content decreased 25% in iron-deficient Arabidopsis seedlings, whereas the ASC levels were not affected. Taken together, these results showed that GSH and ASC supplementation protects Arabidopsis seedlings from iron deficiency, preserving cell redox homeostasis and improving internal iron availability. - Abiotic stresses generally result in an imbalance of cell redox status due to the overproduction of oxidative radicals. This leads, in turn, to an increase in the synthesis of antioxidants such as glutathione (GSH) and ascorbic acid (ASC) and to an increase in the activity of antioxidant enzymes (Noctor and Foyer, 1998). Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), superoxide anion (O2), and hydroxyl radical (OH) can be detoxified by oxidizing GSH andASC. The ASCGSH cycle is a mechanism for removing H2O2. It consists of the enzymes ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and glutathione reductase (Foyer and Noctor, 2011). APX reduces H2O2 to water through the oxidation of ASC, which is subsequently reduced by sequential reactions carried out by the enzymes monodehydroascorbate reductase and DHAR using GSH. Finally, glutathione reductase regenerates GSH from its oxidized form, GSSG, allowing the cycle to continue. Because of its high cell concentrations, GSH and ASC act as ROS scavengers, keeping cell redox homeostasis under control. Many other primary and secondary metabolites may play a similar role; however, GSH and ASC differ from most because of the following characteristics: (i) there are specific enzymes that link GSH and ASC with H2O2 metabolism, (ii) the stability of the corresponding oxidized forms, and (iii) the ability to be recycled to reduced forms through a powerful enzymatic system that depends on the electron transport molecule NAD(P)H (Foyer and Noctor, 2011). Although it has been determined that GSH and ASC can respond in a compensatory way to the cell redox imbalance, it has also been demonstrated that these two compounds have Abbreviations: A, absorbance; ANOVA, analysis of variance; APX, ascorbate peroxidase; ASC, ascorbic acid; CAT, catalase; DAB, 3,3-diaminobenzidine; DHAR, dehydroascorbate reductase; Fd, ferredoxin; GRX, glutaredoxin; GSH, glutathione; H2DCF DA, 2,7-dichloro-dihydrofluorescein; ROS, reactive oxygen species; SE, standard error; SOD, superoxide dismutase. The Author [2013]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For permissions, please email: other specific functions, highlighting that they should not be considered merely as antioxidants. Arabidopsis mutants deficient in GSH have shown that GSH plays a critical role in embryo and meristem development (Vernoux etal., 2000; Cairns et al., 2006; Bashandy et al., 2010), while complete deficiency of ASC causes lethality at the seedling stage (Dowdle etal., 2007). GSH is involved in detoxification of xenobiotics and heavy metals, storage and transport of reduced sulfur, regulation of nuclear and plastid gene expression, and pathogen resistance, among others (Mullineaux and Rausch, 2005; Meyer, 2008; Rouhier et al., 2008; Foyer and Noctor, 2009). It has been established that GSH is closely related to glutaredoxins (GRX), enzymes that have a role in protein regulation through glutathiolation. Glutathiolation can protect the protein thiol groups of irreversible inactivation by oxidation and can also regulate the activity in a positive or negative way (Rouhier etal., 2008). It has been determined by in vitro and in vivo experiments that GRX14 and GRX16 are involved in the incorporation of [2Fe-2S] groups into proteins (Bandyopadhyay etal., 2008). ASC participates in the regulation of programmed cell death, flower senescence, and response against pathogen attack, and in the protection of plants exposed to UV, heat, and high light intensity (Linster and Clarke, 2008; Foyer and Noctor, 2011). There are few studies that have evaluated the relationship between nutrient deficiency and antioxidant defence mechanisms in plants. In sunflower, the level of H2O2 increases in the leaves and this correlates with a decrease in the activity of APX, peroxidases, and superoxide dismutase (SOD) as a consequence of low iron availability (Ranieri etal., 2001). In other studies, it was found that GSH and ASC levels were increased in cucumber and sugar beet exposed to conditions of iron deficiency (Zaharieva et al., 1999; Zaharieva and Abada, 2003). In this work, we have presented the results of studies directed to understand the effects of GSH and ASC in Arabidopsis seedlings grown under conditions of iron deficiency. Our results indicated that supplementation with GSH and ASC protect Arabidopsis against the detrimental effect of iron deficiency. GSH and ASC were able to preserve chlorophyll content without increasing internal iron concentration. The activity of GSH and ASC appeared to be mediated through their antioxidant capacity, as the protective effect correlated with decreased levels of ROS and higher activity of APX. Therefore, it is postulated that GSH and ASC treatments contribute towards keeping cell redox homeostasis in plants growing under iron-deficient conditions. Material and methods Plant material, growth conditions and treatments Seedlings from Arabidopsis thaliana ecotype Columbia (Col-0) were used for these experiments. The seeds were surface sterilized in a solution containing 30% (v/v) sodium hypochlorite and 0.1% (v/v) Triton X-100 for 15 min, washed, and kept for 3 d at 4 C in darkness for seed stratification. The seeds were then sown on plates containing nutrient solution composed of 5 mM KNO3, 2 mM Ca(NO3)2, 2 mM MgSO4.7H2O, 2.5 mM KPO4, 70 mM H3BO3, 0.01 M MnCl2, 1 M ZnSO4, 0.5 M CuSO4, 0.2 M Na2MoO4, 10 M NaCl, 0.01M CoCl2, and 50M FeNaEDTA. The nutr (...truncated)